The catalytic subunit of the proteasome is engaged in the entire process of estrogen receptor-regulated transcription

Abstract

The ubiquitin-proteasome system plays an important role in a variety of cellular functions by means of its proteolytic activity. Interestingly, recent studies have indicated that the proteasome components are also integral parts of transcription complexes. In genome-wide screening for steroid receptor coactivator (SRC)-interacting proteins using yeast two-hybrid system, we found that the 20S proteasome beta subunit LMP2 (Low Molecular mass Polypeptide 2) interacts directly with the SRC coactivators. We showed that LMP2 is required for estrogen receptor (ER)-mediated gene transcription and for estrogen-stimulated cell cycle progression. We found that LMP2-associated proteasome is recruited to the entire sequence of ER target genes, implicating a role for the proteasome in both transcription initiation and elongation. We demonstrated that the recruitment of LMP2 by SRC coactivators is necessary for cyclic association of ER-regulated transcription complexes on ER targets. These results revealed a mechanism by which the proteasome machinery is recruited in ER-mediated gene transcription. Our experiments also provided evidence implicating SRC coactivators in gene transcription elongation.

Figure 1

Identification of LMP2 as an SRC-interacting protein. (A) Schematic representation of SRC proteins and their domains and deletions. (B) Yeast two-hybrid screening. (a) Negative control: cotransformation of yeast AH109 cells with pGBKT7-Lam and GADT7-T plasmids; (b) positive control: cotransformation of yeast AH109 cells with pGBKT7-53 and GADT7-T plasmids; (c) and (d) positive clones from the primary selection of mammary library using pGBKT7-SRC-1-N or pGBKT7-GRIP1-N, respectively; (e) and (f) confirmation of the interaction between LMP2 and SRC-1 or between LMP2 and GRIP1 by cotransformation of the yeast AH109 cells with isolated pACT2-LMP2 and pGBKT7-SRC-1-N or pGBKT7-GRIP1-N, respectively. (C) GST pull-down assays with ³⁵S-labeled full-length SRC coactivators or their C- or N-terminal mutants and GST-LMP2 fusion protein. (D) Co-immunoprecipitation experiments. ECC-1 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) to 95% confluence and cellular nuclear extracts were prepared and immunoprecipitated with antibodies against LMP2. The immunoprecipitated materials were subjected to Western blotting analysis with antibodies against SRC-1, GRIP1, or AIB1. (E) GST pull-down assays with ³⁵S-labeled full-length LMP2 and GST-ERα fusion protein. SRC-1 is included as a positive control.

Figure 2

LMP2 enhances ER-mediated transcription. (A) LMP2 overexpression did not result in any significant changes in SRC protein expression. ECC-1 cells were transfected with an empty vector or an LMP2 expression vector. Seventy-two hours after transfection, cellular extracts were prepared and Western blotting analysis was carried out to analyze the expression of SRC-1, GRIP1, and AIB1. (B) LMP2 enhanced ER-mediated transcription. ECC-1 cells were transfected with cyclin D1 promoter-driven luciferase construct (cyclin D1-luc) or ERE-tk-luciferase (ERE-luc) reporter plasmid together with pcDNA3.1-LMP2 or pSUPER-LMP2-siRNA constructs. Forty-eight hours after transfection, cells were treated with 100 nM of E2 and reporter activity was measured. The firefly luciferase data for each sample were normalized to Renilla luciferase activity. Each bar represents the mean±s.d. for triplicate experiments. (C) LMP2 enhanced the expression of endogenous ER target genes. The expression of ER target genes cyclin D1 and pS2 was measured by real-time RT–PCR in ECC-1 cells transfected with either LMP2 expression construct or LMP2 siRNA construct. Each bar represents the mean±s.d. for triplicate experiments. (D) Collaboration of LMP2 and SRC coactivators in enhancement of ER target transcription. ECC-1 cells were transfected with the ERE-luciferase reporter plasmid together with pcDNA3.1-LMP2 or SRC expression plasmids or pSUPER vector-based RNAi constructs. Forty-eight hours after transfection, cells were treated with 100 nM of E2 and the cells were harvested and firefly luciferase and Renilla activities were measured. Each bar represents the mean±s.d. for triplicate experiments. 3xp160 RNAi means transfection of a combination of three p160 RNAi constructs for silencing the expression of all SRC-1, GRIP1, and AIB1. (E) Western blotting analysis of the protein expression in ECC-1 cells that were transfected with the indicated plasmids. Transfection efficiency was monitored by cotransfection with the E. coli lacZ construct.

Figure 3

LMP2 is recruited on the entire sequence of pS2 gene. (A) A map of the pS2 gene showing the regions that were amplified by PCR in ChIP experiments. (B) The recruitment of LMP2, ERα, and SRC coactivators on promoter as well as on other regions of pS2 gene. EEC-1 cells were grown in the absence of estrogen for 3 days and treated with E2 for 45 min. The cells were then collected and ChIP experiments were performed with different primer pairs described in Materials and methods. (C) The size range of the DNA fragments used in ChIP experiments. (D) The recruitment of ERα, SRC-1, LMP2, Elongin A, and pol II on pS2 gene promoter. EEC-1 cells were grown in the absence of estrogen for 3 days and treated with E2 for 45 min. The cells were then collected and ChIP and ChIP Re-IP experiments were performed with primer a described in Materials and methods.

Figure 4

The recruitment of LMP2, Elongin A, and pol II on pS2 gene. EEC-1 cells were grown in the absence of estrogen for 3 days and treated with E2 for 45 min. The cells were then collected and ChIP and ChIP Re-IP experiments were performed with different primer pairs described in Materials and methods.

Figure 5

The dynamic recruitment of ERα, SRC-1, LMP2, Elongin A, and pol II on pS2 gene. (A) EEC-1 cells were transfected with RNAi constructs to silence the expression of SRC-1, GRIP1, and AIB1 (3xp160 RNAi) or the expression of LMP2. Forty-eight hours after transfection, the cells were transfected again with a mouse LMP2 (mLMP2) expression construct. The cells then were switched to estrogen-depleted media for 3 days and treated with E2 for different periods of time. The cells were then collected at a 5-min interval and quantitative ChIP PCR was performed with primer a (for ERα, SRC-1, LMP2, and pol II) or primer b (for Elongin A). (B) Western blotting analysis of the protein expression in ECC-1 cells treated as above. (C) ECC-1 cells treated as above were transfected with ERE-luc reporter. After addition of E2 for 12 h, the cells were harvested and reporter activity was measured. The firefly luciferase data for each sample were normalized to Renilla luciferase activity. Each bar represents the mean±s.d. for triplicate experiments.

Figure 6

The association of LMP2 with proteasome on pS2 gene. (A) Inhibition of ER-mediated transactivation by proteasome inhibitor MG132 in ECC-1 cells. ECC-1 cells were seeded in DMEM medium supplemented with 10% charcoal-dextran-stripped FBS and transfected with either vector or pcDNA3.1-LMP2 together with the reporter construct ERE-luc. Forty-eight hours after transfection, cells were treated with E2 in the absence or presence of proteasome inhibitor MG132 for 12 h and luciferase activities were assayed. Each bar represents the mean±s.d. for triplicate experiments. (B) Co-recruitment of LMP2 with Rpt6 and pol II on pS2 gene. ECC-1 cells were grown in phenol red-free DMEM medium supplemented with 10% charcoal-dextran-stripped FBS and transfected with the LMP2 siRNA construct. Forty-eight hours after transfection, the cells were left untreated or treated with 100 nM of E2 for 45 min. ChIP assays were performed using specific antibodies against Rpt6, pol II, and LMP2 and with primers for different regions of the pS2 gene. (C) Western blotting analysis of the expression of LMP2, Rpt6, and pol II.

Figure 7

LMP2 promotes estrogen-stimulated cell cycle progression. ECC-1 cells were cotransfected with GFP and LMP2 expression constructs or with GFP and LMP2 RNAi constructs and were grown in phenol red-free DMEM supplemented with 10% charcoal-dextran-stripped FBS for 3 days before treatment with 100 nM of E2 for another 12 h. Cells were then collected for cell flow cytometry analysis. The expression of LMP2 was analyzed by Western blotting and is shown in the lower right.